1. Field of the Invention
The present invention relates generally to the field of molecular biology and immunology. More particularly, it concerns methods for high-throughput isolation cDNAs encoding immune cell receptors and antibodies.
2. Description of Related Art
There is a need to identify the expression of two or more transcripts from individual cells at high throughput. In particular, for numerous biotechnology and medical applications it is important to identify and sequence the gene pairs encoding the two chains comprising adaptive immune receptors from individual cells at a very high throughput in order to accurately determine the complete repertoires of immune receptors expressed in patients or in laboratory animals. Immune receptors expressed by B and T lymphocytes are encoded respectively by the VH and VL antibody genes and by TCR α/β or γ/δ chain genes. Humans have many tens of thousands or millions of distinct B and T lymphocytes classified into different subsets based on the expression of surface markers (CD proteins) and transcription factors (e.g., FoxP3 in the Treg T lymphocyte subset). High-throughput DNA sequencing technologies have been used to determine the repertoires of VH or VL chains or, alternatively, of TCR α and β in lymphocyte subsets of relevance to particular disease states or, more generally, to study the function of the adaptive immune system (Wu et al., 2011). Immunology researchers have an especially great need for high throughput analysis of multiple transcripts at once.
Currently available methods for immune repertoire sequencing involve mRNA isolation from a cell population of interest, e.g., memory B-cells or plasma cells from bone marrow, followed by RT-PCR in bulk to synthesize cDNA for high-throughput DNA sequencing (Reddy et al., 2010; Krause et al., 2011). However, heavy and light antibody chains (or α and β T-cell receptors) are encoded on separate mRNA strands and must be sequenced separately. Thus, these available methods have potential to unveil the entire heavy and light chain immune repertoires individually, but cannot yet resolve heavy and light chain pairings at high throughput. Without multiple-transcript analysis at the single-cell level to collect heavy and light chain pairing data, the full adaptive immune receptor, which includes both chains, cannot be sequenced or reconstructed and expressed for further study.